Rapid, Radiolabeled Macrophage Culture Method for ... - Europe PMC

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1982, p. 26-32 0066-4804/82/010026-07$02.00/0

Vol. 21, No. 1

Rapid, Radiolabeled Macrophage Culture Method for Detection of Dapsone-Resistant Mycobacterium leprae INDIRA NATH,l* H. K. PRASAD,'t M. SATHISH,1 SREEVATSA,2 K. V. DESIKAN,2 p. S. SESHADRI,3 AND C. G. S. IYER3 Department of Pathology, All India Institute of Medical Sciences, New Delhi 110029,1 Central JALMA Institute for Leprosy, Agra 282001,2 Central Leprosy Training Research Institute, Chingleput 603001,3 India Received 7 October 1981/Accepted 23 October 1981

Mycobacterium leprae cells extracted from the skin biopsies of 14 bacilliferous lepromatous patients were maintained in human-murine macrophage cultures for 3 weeks in the presence of [3H]thymidine and DDS (4,4'-diaminodiphenyl sulfone). All cultures except one containing freshly extracted viable bacilli showed significant incorporation of [3HJthymidine as compared to control cultures containing heat-killed bacilli of the corresponding strain. Six susceptible strains of M. leprae obtained from untreated, freshly diagnosed patients showed significant inhibition of the uptake of the radiolabel in the presence of 3 and 10 ng of DDS per ml per culture. Eight strains of M. leprae obtained from patients clinically suspected of DDS resistance were tested in a similar manner. These strains were also concurrently inoculated in the footpads of mice given orally 10-2, 10-3, and 10-4 g of DDS per 100 g of body weight for 9 months. Concordant results were obtained by both methods: five strains were found to be resistant, one was susceptible, and one was partially resistant. Strain VIII did not incorporate [3H]thymidine in the macrophage cultures and proved to be resistant in the mouse footpad. The macrophage culture system provides a sensitive, rapid screening method for the early diagnosis of DDS resistance.

of resistance in the endemic areas using monotherapy. After the reports of radiolabeling of mycobacteria (15) and, in particular, M. leprae (1, 4, 12, 31), our laboratory was able to confirm the uptake of [3H]thymidine by more than 50 M. leprae strains resident in human (20) and murine (25) macrophages. Differentiated macrophages maintained in vitro provide a suitable environment for the short-term maintenance of M. leprae, as indicated by morphological criteria (24) or, in our hands, by the selective uptake of [3H]thymidine by the bacilli (20, 25). The incorporation of the radiolabel appears to be maximal by the third week in culture, and the increase in radioactive counts is cumulative. That the incorporation is related to the viability of M. leprae is indicated by the significant increase in counts in cultures containing freshly extracted bacilli as compared to cultures with the corresponding heat-killed strain. The present study was designed to assess the utility of this in vitro system for the identification of DDS resistance in clinically suspected patients. To test the validity of the system, studies were undertaken in three independent centers in India. The patients were screened in the Central Leprosy Training Research Institute, Chingelput, and the skin biopsies were air-

Mycobacterium leprae continues to evade in vitro cultivation by conventional methods. The problems related to the study of the pathogenesis of leprosy, the effect of antileprosy drugs (9, 13, 22, 27), and the diagnosis of resistant strains (16, 19) were partly overcome with the development of the mouse footpad model as a useful tool in various laboratories (3, 21, 26). For decades, the most suitable and mainly used drug in leprosy has been dapsone (4,4'-diaminodiphenyl sulfone [DDS]). Since the first report of Pettit and Rees in 1964 (19), DDS resistance has emerged as a major cause for concern in many countries of the world (6, 10, 16, 18). Moreover, the anticipated development of primary resistance in endemic areas harboring bacilliferous, infectious patients with DDS-resistant M. leprae has grave economic and public health implications (32). The mouse footpad model, which has served as the main test system for the last 2 decades, requires 6 to 9 months for the full expression of bacterial multiplication. Thus, a rapid and reliable method is required for the early detection of drug-resistant strains to institute prompt treatment and assess the prevalence t Present address: National Hansen's Disease Center, U.S. Public Health Service Hospital, Carville, LA 70721.

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RAPID DETECTION OF DDS-RESISTANT M. LEPRAE

freighted on wet ice to the All India Institute of Medical Sciences, Delhi, for screening in the macrophage cultures, and to the Central JALMA Institute, Agra, for inoculation in the mouse footpad. The results obtained in the macrophage c} 'tures in 3 weeks were compared with those obtained in the mouse model 9 months later. Data from six M. leprae strains obtained from the same clinic from untreated lepromatous leprosy patients tested in an identical manner in the macrophage culture system are also included. MATERIALS AND METHODS Padents. Eight patients with bacilliferous lepromatous leprosy attending the clinics of the Central Leprosy Training and Research Institute, Chingelput, from June to September 1979 were clinically suspected of DDS resistance on the basis of (i) the development of newer lesions and (ii) increase in acid-fast bacilli in skin slit smears (23), as indicated by bacteriological and morphological indices. They had been on intermittent or regular therapy with DDS, ranging in dosage from 25 to 300 mg per week for 4 to 10 years (Table 1). Routine excision skin biopsies of these patients were taken under sterile conditions and air freighted on ice to the respective laboratories for testing in the macrophage assay system and the mouse footpad model. Biopsies from six untreated lepromatous leprosy patients were also tested in a similar manner in the macrophage system but not in the mouse footpad. Extrction of M. kpme. In brief, M. leprae bacteria were extracted under sterile conditions by tissue homogenization of biopsies after the removal of the epidermis and subcutaneous fat (21). The bacilli were suspended in 0.9%o saline and centrifuged at 500 rpm for 10 min to remove coarse tissue particles. The supernatant was removed and centrifuged at 2,500 rpm for 40 min. The pellet containing bacilli was washed once with saline and subsequently suspended in RPMI 1640 (GIBCO Biocult). Samples were diluted in 1% albumin saline and counted by the method of Shepard and McRae (28).

27

Before inoculation in cultures, M. leprae extracts checked for the absence of cultivable contaminants by plating on nutrient agar for 48 h and Lowenstein-Jensen medium for 8 weeks. None of the extracts used showed growth of contaminants. M. leprae (106) were inoculated into each Leighton tube within 48 to 72 h after the removal of the biopsy. Control cultures were inoculated with heat-killed bacilli of the corresponding strain. The number of bacilli extracted from the biopsies ranged from 3 x 107 to 4 x 108. Macrophage cultures. Macrophage cultures were set up as described earlier (20, 25). In brief, adherent mononuclear cells from the peripheral blood of normal volunteers and peritoneal resident macrophages from BALB/c mice were cultured in Leighton tubes containing RPMI 1640 supplemented with 50% autologous plasma and 30%6 fetal calf serum (GIBCO Biocult), respectively. After incubation at 37C for 16 to 18 h, the nonadherent cells were removed and RPMI 1640 with 50o AB serum or 30% fetal calf serum was added to human and murine cultures, respectively. Macrophages showed morphological differentiation by S to 6 days in the human and 24 to 48 h in the murine cultures. M. leprae were inoculated at this stage. After the removal of unphagocytosed bacilli at 18 h, the medium was replaced and supplemented with 100 pl of medium containing 1 ,Ci of [methyl-3H]thymidine (Amersham Radiochemical Centre; specific activity, 42 Ci/mmol) and 3 and 10 ng of DDS. The medium, radiolabel, and drugs were replaced every 4 to 5 days. After 14 days of incubation, the cultures were harvested on fiber glass disks. Macrophages were stripped with a rubber policeman after treatment with 10%o Triton X-100 (Sigma Chemical Co.). The disks were washed serially at 4°C with saline containing 1 mg of unlabeled thymidine per ml, 5% trichloroacetic acid, and methanol. The dried disks were placed in scintillant mixture and counted in a 1B scintillation

were

counter.

Using the

same

batch of macrophages, five repli-

cates each were put up as follows: (i) macrophage

cultures alone, (ii) macrophages plus freshly extracted "viable" M. leprae, (iii) macrophages plus heat-killed

TABLE 1. Clinical details of lepromatous leprosy patients suspected of DDS resistance Pattern of treatment with DDS (25-100 mg daily) Patient Age Sx Duration of B.I.a M.I.b no. disease (yr) (yr) I 1 30 M 27 DDS, irregular, clofazimine, steroids during 2.83 NDc reactional states. II 2 42 M 10 Irregular, discontinued for 7 yr. Restarted for 1 yr. 2.66 4 III 3 40 M 21 Regular for 12 yr, discontinued for 7 yr, restarted 3.67 ND for 1 yr. IV 4 30 M 10 Irregular. Regular for 1 yr. 3.33 0.66 5 V 20 F 4 Regularfor 2.5 yr. 2.83 1.42 VI 6 40 M 15 Regular. 2.16 0.1 VII 7 35 M 10 Irregular. ND ND VIII 8 42 M 28 Irregular, interrupted, clinical relapse. ND ND I B.I., Bacteriological index. The arithmetic mean of the logarithmic index of bacilli from slit smears of six sites (23). b M.I., Morphological index. Percentage of solid-staining acid-fast bacilli in skin smears. Stained by ZiehlNeelson method. c ND, Not done. Strain

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NATH ET AL.

M. leprae of the same strain, (iv) (ii) + 10 ng of DDS, and (v) (ii) plus 3 ng of DDS. Mean counts per minute ± standard error of three replicate cultures were calculated. The percent inhibition in the presence of DDS was calculated as follows: percent incorporation = 100 x [(Mean cpm of macrophage cultures + live M. leprae + DDS) - (Mean cpm of macrophage cultures + autoclaved M. leprae)]l [(Mean cpm of macrophage cultures + live M. leprae) - (Mean cpm of macrophage cultures + autoclaved M. leprae)]. Percent inhibition = 100 - percent incor-

poration. DDS was donated by K. Desikan, Central JALMA Institute. A stock solution of DDS (1 mg/ml) was initially dissolved in 1 N HCI and subsequently diluted in RPMI 1640 before use. Statistical analysis was done by the Mann-Whitney two-tailed test and Kruskal Wallis one-way analysis of variance (30). Mouse footpad studies. M. leprae (5 X 103 to 104) from the corresponding half of the biopsy was inoculated into the left footpads of normal BALB/c mice as described earlier (3). DDS was administered to the animals orally, and they were divided into the following batches: (i) no treatment, (ii) 10-2 g of DDS per 100 g, (iii) 10' g of DDS per 100 g, and (iv) 10-4 g of DDS per 100 g. From 6 to 9 months two mice from each batch were killed at monthly intervals. M. leprae bacteria were extracted from the footpads by homogenization in glass homogenizers, and the bacillary yield was counted as described above. The highest yield of bacilli in each group is expressed below as logarithmic values.

RESULTS Fourteen bacilliferous leprosy patients were included in the study, of whom six were seen for the first time and gave no history of treatment and eight were clinically suspected of DDS resistance. As may be observed in Fig. 1, M. leprae strains IX to XIV obtained from the six untreated lepromatous leprosy individuals showed significant incorporation of [3H]thymidine in cultures containing viable bacilli as compared to control cultures containing the corresponding heat-killed strain. In the presence of 3 and 10 ng of DDS per ml per culture, significant graded inhibition of the uptake of the radiolabel was observed (P < 0.05 to < 0.001), which was in conformity with our earlier studies (submitted for publication). Thus, these strains were considered susceptible to the drug. Suspected DDSresistant strains of M. leprae were also tested at these concentrations of DDS. Table 1 gives the details of therapy and the quantum and morphological characteristics of the bacilli in smears of skin scrapings from the eight patients clinically suspected of DDS resistance. Except for one individual, all had been on monotherapy with DDS for several years. During the year of study, all patients had developed

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DDS Concentration (ng / ml) FIG. 1. Effect of 3 and 10 ng of DDS per ml per culture on [3H]thymidine incorporation of six strains of M. Ieprae derived from untreated, freshly diagnosed patients of lepromatous leprosy and maintained in macrophage cultures for 3 weeks. Graded and significant inhibition of the uptake of the radiolabel was noted in drug-treated cultures of all of the strains (P < 0.05 to < 0.001). Symbols: U, macrophages with heatkilled M. Ieprae; O, macrophages with corresponding strain of viable M. Ieprae; ~, macrophages with corresponding strain of viable M. Ieprae plus 3 ng of DDS per ml; IIID, macrophages with corresponding strain of viable M. Ieprae plus 10 ng of DDS per ml. Mean counts per minute ± standard error of three replicate cultures. new bacilliferous lesions while on treatment with a daily dose of 100 mg of DDS. Figure 2 depicts the integrated data obtained on the pattern of growth in eight human-derived M. leprae strains as assessed by (A) [3H]thymidine incorporation in macrophage cultures (histograms) and (B) bacillary counts in mouse footpads (graphs). The results obtained from the in vitro assay by 3 weeks are compared with the mouse inoculation studies at the end of 9 months. DDS was present in concentrations of 10 and 3 ng per ml per culture for the duration of the culture period, whereas in the mouse footpad model, batches of animals were given orally 10 gover a 9m0a,c10r,pandl0-2r g of DDS pere month period. Effect of DDS on [3Hlthymidine incorporation in M. leprae within macrophages. Six strains were tested in the human (III, IV, V, VI, VII, and VIII) and two were tested in the murine (I,

VOL. 21, 1982

RAPID DETECTION OF DDS-RESISTANT M. LEPRAE

29

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DDS FIG. 2. Effect of various concentrations of DDS on eight strains of M. leprae derived from lepromatous patients clinically suspected of DDS resistance. Each strain was assayed concurrently by: (A) in vitro macrophage cultures incubated with [3H]thymidine (histograms) and (b) in vivo multiplication of bacilli in the mouse footpad model (graphs). The strains are designated by roman numerals, and the dose of DDS used in the two methods is indicated against the respective assay. The viability of M. leprae in the presence of DDS was assessed in the macrophage cultures by mean counts per minute ± standard error for three replicate cultures and as log1o of maximal bacillary counts in footpads of two mice. P value, Mann-Whitney (two-tailed) test. NS, Nonsignificant; NEG, bacilli not detected. Symbols: *, cultures with heat-killed bacilli; O, cultures with viable bacilli of corresponding strain; U, cultures with viable bacilli of corresponding strain plus 3 ng of DDS; E, cultures with viable bacilli of corresponding strain plus 10 ng of DDS.

II) macrophage cultures. All except strain VIII had shown significant incorporation of [3H]thymidine ranging from 191 to 466% (P < 0.05 to < 0.001) in cultures containing viable freshly extracted M. leprae as compared to cultures containing heat-killed bacilli of the corresponding strain (Table 2). It may be observed that individual cultures varied in the degree of [3H]thymidine incorporation and in the base-line counts per minute of control cultures of macrophages containing heat-killed bacilli. The murine macrophage cultures showed higher counts per minute than did human cultures (Fig. 2).

The results obtained in the macrophage cultures are shown in Table 2 and as histograms in Fig. 2. Strain I, maintained in murine macrophages, showed significant inhibition of [3H]thymidine in cultures treated with 10 and 3 ng of DDS per ml as compared to untreated cultures with viable freshly extracted bacilli. The counts per minute of drug-treated cultures at both concentrations reached base-line values obtained in control cultures with heat-killed bacilli (Fig. 2). The percent inhibition of [3H]thymidine uptake was 77 and 88 at 3 and 10 ng of DDS per ml, respectively (P < 0.05; Table 2).

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NATH ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

TABLE 2. Pattern of [3H]thymidine incorporation in drug-free and drug-treated macrophage cultures containing freshly extracted viable bacilli from clinically suspected DDS-resistant leprosy patients

Strain no.

Patient

% Incorporation in drug-free cultures

% Inhibition and probabilitya in cultures treated with DDS at (ng/ml):

Diagnosis by:

P In vitro 10

3

macrophage assay

footpad assay

I 1 360